The long-term goal of this research program is to increase our understanding of how cellular transport and metabolism influence the oral bioavailability of dietary flavonoids, a large class of compounds that has been implicated to play a major role in the prevention of human diseases, in particular cardiovascular disease and cancer. In Specific Aim 1 we will determine the interrelationships between SGLT1 and MRP2, including mechanisms involved, in the enterocyte absorption of flavonoid glycosides and the tea flavonoids, two main classes of dietary flavonoids. These studies will be undertaken in SGLT1- and MRP2-transfected cells and in the human intestinal absorption model Caco-2. The role of the potentially most important transporter, i.e. MRP2, will be directly examined in vivo in the MRP2-deficient Tr- rat. In Specific Aim 2 we will investigate the interrelationships between CYPs, UGTs and SULTs, including the identification of the major isoforms involved, in the hepatic as well as intestinal metabolism of flavonoids. This will be done in microsomes as well as in intact cells, e.g. fresh human hepatocytes. These experiments will allow us to establish the major pathway(s) of metabolism of the flavonoids. In addition, autoinduction of flavonoid metabolism will be examined, mainly focusing on CYPs and UGTs. The importance of the UGT family of enzymes will be directly examined in vivo in the genetically deficient Gunn rat. In Specific Aim 3 we will determine the role and mechanisms of a) bacterial- and b) peroxidase-mediated catabolism of flavonoids, including covalent binding to protein. The experiments in a) will be conducted in gnotobiotic compared to normal rats as well as in samples from an in vivo human study. Complementary in vitro studies will include the identification of the bacterial pathway leading from quercetin to CO2 formation. The experiments in b) will be conducted in vitro, using pure enzymes and subcellular fractions, and then in intact cell systems in which production of reactive oxygen species as well as glutathione levels can be manipulated. Structure identification of metabolites as well as elucidation of covalent binding will be critical factors. The findings from the proposed studies should help us understand the bioavailability of the flavonoids, facilitating optimization of the chemopreventive utility of these natural or synthetic compounds.